Controllable Self-Assembly of Nanoparticles for Specific Delivery of Multiple Therapeutic Molecules to Cancer Cells Using RNA Nanotechnology

Abstract

By utilizing RNA nanotechnology, we engineered both therapeutic siRNA and a receptor-binding RNA aptamer into individual pRNAs of phi29's motor. The RNA building block harboring siRNA or other therapeutic molecules was fabricated subsequently into a trimer through the interaction of engineered right and left interlocking RNA loops. The incubation of the protein-free nanoscale particles containing the receptor-binding aptamer or other ligands resulted in the binding and coentry of the trivalent therapeutic particles into cells, subsequently modulating the apoptosis of cancer cells and leukemia model lymphocytes in cell culture and animal trials. The use of such antigenicityfree 20-40 nm particles holds promise for the repeated long-term treatment of chronic diseases.

Construction of folate-conjugated pRNA of bacteriophage phi29 DNA packaging motor for delivery of chimeric siRNA to nasopharyngeal carcinoma cells

“…A chimeric pRNA/siRNA monomer was constructed by replacing the double-stranded helical region of pRNA with siRNA sequences without affecting the gene silencing function and the pRNA secondary structure. 21,22 The deliverable folate containing nanoparticles was conjugated by mixing equal molar amounts of folate-pRNA (B-a 0 ) with a chimeric pRNA/siRNA (A-b 0 ) via the interaction of the interlocking loops ( Figure 3 Entry of nanoparticles containing both folic-pRNA and siRNA chimera to nasopharyngeal carcinoma cells To determine whether the folate moiety on the chimeric pRNA dimer could mediate the entry of the complex into KB cells, a chimeric siRNA complex was constructed for specific gene silencing. RNA dimer (1.75 mM), containing both folate and siRNA against firefly luciferase, was incubated with, rather than transfected into, KB cells.…”

“…16,21,22 In this study, a chimeric pRNA/siRNA targeting firefly or renilla luciferase was constructed, and the silencing efficiency was tested by transient transfection. The chimeric siRNA construct suppressed its target gene specifically and efficiently as demonstrated by a Dual reporter assay, in which the expression To determine whether the knockdown effects shown above were siRNA-specific, chimeric pRNA/siRNA monomers or dimers were treated with cell lysate or recombinant purified Dicer ( Figure 5), which is known for its unique function in processing long doublestranded RNA into 22-bp siRNA.…”

“…[18][19][20] Recently, we found that phi29 pRNA can be used as a carrier for the construction of RNA nanoparticles to deliver therapeutic RNAs such as siRNAs and/or ribozymes to specific cancer cells. 12,21,22 In this report, we described the methods for the synthesis and purification of folate-adenosine 5 0 -monophosphate (AMP), the procedure for the construction of folatepRNA, and the test of the purity and function of the folate-containing products. The potential of siRNA in gene therapy mediated by folate was also investigated in animal trials.…”

Engineered RNA Nanodesigns for Applications in RNA Nanotechnology

“…non-RNA nature (proteins, small targeting molecules, gold NP, etc) [93][94][95][96], discussed in detail in Functionalization and Modification, below. The second class encompasses tectonic units that can be split into structural motifs and interacting motifs possessing discrete secondary and tertiary structures which can be combined to rationally produce self-assembling nanoparticles with predictable, multi-dimensional structures in vivo or in vitro.…”

“…This combination of specific, programmable supramolecular structures with diverse functional units in a single, entirely RNA compound, allows precise positioning of different functional modules in three-dimensional space. This, for example, can provide an avenue to address the vital problems of stability, targeting, and multivalency in nanomedicine [95,102,103,107], and can form the basis of preprogrammed arrays [43], mediate the growth and distribution of other NPs [94,108], or form patterned scaffolds for tissue engineering or other applications [47].…”